Purpose:
Fibrosis can complicate several retinal diseases, including epiretinal membranes, proliferative vitreo-retinopathy, and age-related macular degeneration. MicroRNA-192 is a known mediator of transforming growth factor-β1 (TGF-β1) pro-fibrotic effects in kidney diseases. However, the role of miR-192 in Epithelial-Mesenchymal Transition (EMT), a key process in tissue repair and fibrosis, has not been yet investigated. The purpose of this study was to assess the role of miR-192 in TGF-β1-induced EMT of RPE cells.

Methods:
ARPE-19 cells were treated with TGF-β1 (2.5, 5, 10 ng/mL), and RNA was extracted at 72 hours. miR-192 expression was quantitated by qRT-PCR. Following, ARPE-19 cells were treated with TGF-β1, and pretreated an AKT-specific inhibitor MK-2206. Expression of epithelial (E-Cadherin and ZO-1) and mesenchymal (Smooth Muscle Actin and Vimentin) EMT markers, and phosphorylated AKT, were assessed by Immunostaining and Western Blot. In addition, cells were transfected with miR-192 mimic, and EMT markers and ratio of activated AKT were assessed by Western Blot. Acetylation of ETS-1 transcription factor, which binds and represses miR-192 gene, was assessed by Immunoprecipitation. All experiments were performed 3 times, in duplicates. Statistical analyses were performed by Student’s t-tests or ANOVA followed by Tukey’s post hoc test. A p value < 0.05 was considered statistically significant.

Results:
miR-192 expression was significantly upregulated 2-fold after treatment with 5 and 10 ng/mL of TGF-β1 (p=0.011 and p=0.015, respectively). Treatment with TGF-β1 down-regulated epithelial markers, up-regulated Mesenchymal markers, and increased phosphorylation of AKT. Pre-treatment with MK-2206 significantly abrogated TGF-β1 induced-EMT and arrested phosphorylation of AKT kinases. Transfection with miR-192 mimic significantly increased the expression of Vimentin and Smooth Muscle Actin, and phosphorylation of AKT kinases. In addition, the ratio of acetylated ETS-1 was significantly increased after treatment with TGF-β1, and was correlated to increased mir-192 expression.

Conclusions:
miR-192 can contribute to EMT in ARPE-19 cells by activation of AKT kinases and inducing the expression of Mesenchymal EMT markers. This suggests that miR-192 may have a role in the development of intraocular fibrosis, and may be a potential therapeutic target for retinal and macular diseases.